Dominique Fagot

Direct role of human dermal fibroblasts and indirect participation of epidermal keratinocytes in MMP-1 production after UV-B irradiation.

Abstract. In vivo, matrix metalloproteinases are produced in response to ultraviolet B (UV-B) irradiation and are considered to be involved in connective tissue alterations observed in photoaging. The respective roles of keratinocytes and fibroblasts in UV-B-induced MMP-1 production were investigated in monolayer cultures of keratinocytes and fibroblasts as well as in an epidermis model reconstructed in vitro. In contrast to fibroblasts, which secreted MMP-1 in response to UV-B irradiation, no accumulation of MMP-1 was observed after UV-B irradiation of keratinocytes. However, culture medium from UV-B-irradiated keratinocytes, which showed an increase in IL-1α and IL-6, induced MMP-1 production by human fibroblasts, suggesting that UV-B irradiation modulates MMP-1 production via both direct and indirect mechanisms.

Matrix Metalloproteinase-1 Production Observed After Solar-Simulated Radiation Exposure is Assumed by Dermal Fibroblasts but Involves a Paracrine Activation Through Epidermal Keratinocytes¶

Chronic exposure of human skin to solar UV radiation leads to serious dermal damages, a hallmark of photoaging. In vivo, acute UV radiation has been shown previously to induce various matrix‐degrading proteases. Among them, matrix metalloproteinase‐1 (MMP‐1) has been suggested to be involved in skin photodamage. The purpose of this study was to investigate the effects of solar‐simulated radiation (SSR) on MMP‐1 production in normal human skin cells. SSR exposure of human skin reconstructed in vitro comprising both a differentiated epidermis and a fibroblast‐populated dermal equivalent led to an increase in MMP‐1 production, which was abolished when epidermis was removed immediately after SSR exposure. In addition, SSR exposure of differentiated keratinocytes grown on an acellular collagen gel did not induce MMP‐1 production. Experiments on cell cultures grown on plastic confirmed that keratinocytes failed, in contrast with fibroblasts, to produce MMP‐1 in response to SSR exposure. However, when conditioned medium from SSR‐exposed keratinocytes was added to human fibroblasts in culture, MMP‐1 production was induced. Altogether, these data show that MMP‐1 production observed after SSR exposure involved the release of soluble epidermal factors, which could modulate its production by dermal fibroblasts.

Multiphoton microscopy of engineered dermal substitutes: assessment of 3-D collagen matrix remodeling induced by fibroblast contraction

Dermal fibroblasts are responsible for the generation of mechanical forces within their surrounding extracellular matrix and can be potentially targeted by anti-aging ingredients. Investigation of the modulation of fibroblast contraction by these ingredients requires the implementation of three-dimensional in situ imaging methodologies. We use multiphoton microscopy to visualize unstained engineered dermal tissue by combining second-harmonic generation that reveals specifically fibrillar collagen and two-photon excited fluorescence from endogenous cellular chromophores. We study the fibroblast-induced reorganization of the collagen matrix and quantitatively evaluate the effect of Y-27632, a RhoA-kinase inhibitor, on dermal substitute contraction. We observe that collagen fibrils rearrange around fibroblasts with increasing density in control samples, whereas collagen fibrils show no remodeling in the samples containing the RhoA-kinase inhibitor. Moreover, we show that the inhibitory effects are reversible. Our study demonstrates the relevance of multiphoton microscopy to visualize three-dimensional remodeling of the extracellular matrix induced by fibroblast contraction or other processes.

The anti-ageing potential of a new jasmonic acid derivative (LR2412): in vitro evaluation using reconstructed epidermis episkin™

Abstract:  Jasmonic acid is involved in plant wound repair and tissue regeneration, but no study has been reported in human skin. The effect of a jasmonic acid derivative, tetra‐hydro‐jasmonic acid (LR2412, 1 and 10 μm) was investigated on an in vitro reconstructed skin model, Episkin™. Using real time RTQPCR studies, results showed an increase in hyaluronan synthase 2 (HAS2) and hyaluronase synthase 3 (HAS3) expression. Furthermore, an increase in hyaluronic acid (HA) deposits in basal and suprabasal layers of the epidermis was observed. The percentage of positive Ki67 keratinocytes in the basal layer as well as the epidermis thickness were seen to increase. Immunohistochemistry studies showed that the synthesis of late differentiation proteins filaggrin and transglutaminase 1 was not modified. The human epidermis is known to thin with age while HA content has been reported to decrease. These results illustrate the potential of LR2412 in counteracting signs of skin ageing.

Multiphoton microscopy of engineered dermal substitutes: assessment of 3D collagen matrix remodeling induced by fibroblasts contraction

One of the main functions of dermal fibroblasts is the generation of mechanical forces within their surrounding extracellular matrix. Investigating molecules that could modulate fibroblast contraction and act as potent anti aging ingredients requires the development of three-dimensional in situ imaging methodologies for dermal substitute analysis. Here we use multiphoton microscopy in order to investigate the fibroblast-induced collagen matrix reorganization in engineered dermal tissue and to evaluate the effect of Y27632, a RhoA kinase inhibitor on dermal substitutes contraction. We observe that collagen fibrils rearrange around fibroblast with increasing density in control samples, whereas collagen fibrils show no remodeling in the samples containing the RhoA kinase inhibitor. Moreover, when the culture medium containing the inhibitor was replaced with a control medium, the dermal substitutes presented the same 3D reorganization as the control samples, which indicates that the inhibitory effects are reversible. In conclusion, our study demonstrates the relevance of multiphoton microscopy to visualize three-dimensional remodeling of the matrix induced by fibroblast contraction.